1. Field of the Invention
This invention relates to treating disease states in a living patient using oxidized lipoproteins, preferably peroxidized low density lipoproteins (p-LDL). More particularly, it relates to a method for increasing the quantity of oxidized or peroxidized lipoproteins, which includes chylomicrons, chylomicron remnants, very low density lipoproteins, intermediate density lipoproteins, low density lipoproteins and high density lipoproteins; taken up by diseased cells. All of these can serve as a source of oxidized lipoprotein. It also relates to a method and an apparatus for producing and administering effective doses of oxidized lipoproteins into a patient's bloodstream. The invention also relates to novel classes of oxidized lipoproteins which can be used to treat disease states.
2. Prior Art
It is possible to distinguish between people with cancer and healthy individuals without cancer on the basis of H-1 and C-13 NMR spectra. Methods and apparatuses for such diagnosis are described in the following patents issued to Eric T. Fossel the teachings of which are incorporated herein by reference: "Process for the Screening of Cancer Using Nuclear Magnetic Resonance"; U.S. Pat. No. 4,912,050, Mar. 27, 1990 and "Process for the Detection of Cancer Using NMR"; U.S. Pat. No. 4,918,021, Apr. 17, 1990.
In accordance with the aforementioned inventions, it was discovered that the majority of resonances in the non-water H-1 signals and the resonances of the C-13 nuclear magnetic resonance (NMR) spectra of fluid samples from both normal and cancerous patients arise from lipids and small molecules. It was found that comparing the full linewidths at half-height of the water-suppressed proton spectra with standard values provides a statistically reliable cancer diagnosis. In particular, comparing the average full linewidths at half-height of the methyl and methylene group resonance lines with a standard value of 33 Hz provides a basis for classifying persons into classes; with and without cancer. Average values below 33 Hz are taken as indicating the presence of malignancy.
It was further discovered that the major source of error in such diagnoses was hypertriglyceridemia. A method was developed to overcome this difficulty in diagnosis. It was found that C-13 spectra provide a further basis for classifying persons with high triglyceride levels (above 190 mg/dl) into two groups; cancerous or non-cancerous. The sample used to generate the H-1 NMR spectrum can be used to generate a C-13 NMR spectrum. The olefinic region of the spectrum is diagnostic. A ratio of the signal at 128 ppm of resonance frequency to that at 130 ppm was obtained. A ratio of greater than 0.9 indicates that the initial characterization from the proton NMR diagnosis was a false positive. However, a 128/130 ratio of less than 0.9 confirms the positive diagnosis. A complete evaluation comprising analysis of both H-1 and C-13 NMR spectra is more accurate than the proton test alone.
The resonance peak at 128 ppm is due to linoleic acid, an eighteen carbon polyunsaturated fatty acid with two double bonds. It must be supplied via ones' diet as the body cannot synthesize such fatty acids. The resonance peak at 130 ppm is due both to polyunsaturated fatty acids such as linoleic acid and monounsaturated fatty acids such as oleic acid, an eighteen carbon fatty acid produced by the body. The relative height of these peaks is affected by the cancerous state of the patient. Persons with untreated cancer have low levels of linoleic acid in their bodies. This is caused by the oxidation of unsaturated fatty acid chains with linoleic acid being more rapidly peroxidized by hydroxyl free-radicals. Since linoleic acid contains a double bond pair capable of delocalizing an electron when in the radical state, its radical intermediate has a lower free energy than the oleic acid radical. Free-radical induced oxidation therefore results in a decreased proportion of polyunsaturated fatty acids such as linoleic to monounsaturated fatty acids such as oleic acid and consequently of the resonance peak at 128 ppm relative to that at 130 ppm.
Linoleic acid is a constituent of triglycerides, cholesterol esters and phospholipids which are components of low density lipoproteins (LDL), the major carrier of cholesterol in the blood. Much of the cholesterol carried by LDL is esterified, principally to linoleic acid. In addition, LDL is made up of phospholipids and a large core protein of 514 kD, protein B-100. All together, LDL is about 22 nm in diameter and has a mass of approximately 3 million daltons. LDL carries cholesterol to peripheral tissues. LDL receptors in coated pits on cell membranes bind protein B-100 and internalize the LDL particles. LDL is broken down inside the cell, and the LDL receptor is returned to the cell membrane. The cholesterol may be incorporated into the cell membrane or stored in the cell in esterified form. Cells which have enough cholesterol stop producing LDL receptors which have a lifespan of about one day. A more detailed discussion of this and related mechanisms can be found in Stryer, Biochemistry, W. H. Freeman and Company, New York, 1988, pp. 560 ff. The foregoing method for detecting cancer has enabled the monitoring of cell components that are diagnostic for cancer. This screening technique has enabled the present discovery by providing insight into the mechanism of action of the present invention.
It was separately known that the cytokine tumor necrosis factor (TNF), a 17 kD protein, has a number of effects, including in vitro and in vivo tumor cell necrosis. TNF is released from macrophages in response to malignant cells. Nathan, J. Clin. Inves., 1987, vol. 79, pp. 319-26. TNF then causes alteration of plasma lipoprotein lipids by suppressing lipoprotein lipase activity. Beutler et al., J. Exp. Med., 1985, vol. 161, p. 984; Semb et al., J. Biolog. Chem., 1987, vol. 262, pp. 8390-94. It also induces polymorphonuclear neutrophils (PMN) to undergo a respiratory burst resulting in lipid peroxidation. Figari et al., Blood, 1987, vol. 70, pp. 979-84.
In addition to its production in response to cancer, TNF is released in response to a variety of other relatively rare and easily definable disease states including malaria, gram negative endotoxin shock, uncontrolled diabetes, AIDS, and organ rejection. Since the 1980's, when TNF-like activities were first described, several TNFs have been isolated and characterized. TNF-a, the original TNF, is the TNF referred to in this application.
The list of activities attributed to TN is growing rapidly. It has been found to have a wide range of biological activities in addition to anti-tumor activity. These include inhibiting lipoprotein lipase activity (Beutler et al, supra; Semb et al., supra), inhibiting bone marrow differentiation (Degliantoni et al., J. Exp. Med., 1985, vol. 162, p. 1512), and interacting with the regulation of producing other cytokines and their receptors (Dinarello et al, J. Exp. Med. 1986, Vol. 163, p. 1433). Despite its many effects, the mechanism by which TNF exhibits its anti-tumor effect remains a subject of debate.
An important step toward understanding the mechanism was taken in a series of experiments with Meth A sarcoma cells by Palladino et al. J. Immunol., 1987, Vol. 138, pp. 4023-32. They suggested that TNF does not directly assert its anti-tumor effect on Meth A sarcoma cells, but rather requires the mediation of other cells or substances. Recent reports by Palladino implicate polymorphonuclear neutrophils (PMN) as mediators. Figari et al., supra. It was shown that TNF (and, to a much lesser extent, other cytokines) stimulates a respiratory burst in PMN resulting in production of superoxide, O.sub.2.sup.-. Superoxide is a precursor to hydroxyl radicals (.multidot.OH), a highly reactive species which oxidizes many tissue constituents.
It is known that cytotoxicity is associated with oxidized lipids. Peroxide itself is toxic to virtually all cell types. More recently, several workers have focused attention on the cytotoxicity of the polyunsaturated fatty acid peroxidation products.
Unfortunately, in addition to all of its useful activities, TNF produces very toxic side effects. It has, therefore, been difficult to use TNF for fighting cancer in humans. It is desirable to find a chemotherapeutic agent which will target cancer cells preferentially.
A study by Edelson et al. N. Eng. J. of Med., 1987, Vol. 316, pp. 297-303 describes treatment effective for some cutaneous lymphomas and certain leukemias whereby a photosensitizer is added to blood and the blood is irradiated with ultraviolet light. In that study, an aromatic compound, 8-methoxypsoralen, was ingested by a patient. Several hours later blood wa withdrawn and irradiated with ultraviolet light (type A) and then re-infused into the patient. This study, however, does not explain the mechanism of this method of treatment, it simply indicates that it is effective against certain forms of cancer.
The present invention provides several methods for oxidizing lipoproteins for the purpose of treating disease states and photoperoxidation is one of these methods. It is believed, that by adding a photosensitizer such as 8-methoxypsoralen to blood and then irradiating the blood with ultraviolet light, radical intermediates may be produced resulting in generation of hydroxyl radicals. It is believed that these hydroxyl radicals in turn oxidize lipoproteins.